The vertebrate immune response to infection begins with the recognition by the innate immune system of conserved molecular signatures of pathogens, known as PAMPs (Pathogen Associated Molecular Patterns), provoking an immediate and often massive inflammatory response. The innate response holds the pathogen in check, but also plays a crucial role in the generation of acquired immunity. The recognition of PAMPs by the innate system is mediated by a number of receptors, of which the Toll-like Receptors (TLRs) play a prominent role. Unlike the antigen receptors of acquired immunity, the TLRs are encoded by a limited number of germline genes, ten in humans; however, in spite of their small numbers, the TLRs recognize a remarkably wide variety of PAMPs including glycolipids, proteins, and nucleic acids. The molecular basis for the recognition of PAMPs by TLRs is a main interest of my laboratory. In collaboration with Dr. David Davies (LMB, NIDDK), we have expressed mg amounts of the extracellular domain (ECD) of TLR3, and have determined its structure by X-ray crystallography. Double-stranded (ds) RNA, a molecular signature of many viruses, binds and activates TLR3. The structure of TLR3-ECD consists of a solenoid of 23 turns, bent into a horseshoe shape, with a large beta-sheet on the concave surface. The molecule is heavily glycosylated, except that one lateral face of the horseshoe is totally devoid of glycan, and this face was predicted, based on mutational analyses, to be the location for ligand binding. To determine how TLR3 binds its ligand, we first studied the interaction of dsRNA oligos with TLR3-ECD protein in solution. We found that purified TLR3-ECD binds dsRNA specifically via a defined ligand-binding site, with an affinity that increases with buffer acidification and ligand size. TLR3-ECD is monomeric in solution, but it forms dimers when bound to dsRNA. These dimers are stabilized by cooperative interactions between the two TLR3-ECDs in a pair, and multiple TLR3-ECD dimers bind to long dsRNAs. The smallest oligonucleotides that form stable complexes with TLR3-ECD (40-50 bp) are also the smallest dsRNAs that activate TLR3 in cells. Thus, we demonstrated that the minimal TLR3 signaling unit is a ligand-bound dimer of TLR3 molecules. To determine the molecular basis for ligand binding and signaling, we isolated, crystallized, and solved the structure of the TLR3 signaling complex, consisting of two TLR3-ECD molecules bound to one 46 bp dsRNA oligonucleotide. The two TLR3-ECDs in the complex face one another, and are related by two-fold symmetry on opposite sides of the dsRNA molecule. We identified three intermolecular contacts that stabilize the complex. The dsRNA interacts with both an N-terminal and a C-terminal site on the glycan-free surface of each mTLR3-ECD. The C-terminal sites are directly across from each other while the N-terminal sites are outstretched at opposing ends of the linear dsRNA molecule. The length of the complex is 141 , which corresponds well with the minimal dsRNA oligo size required for complex formation. In addition, the two TLR3-ECD molecules of the dimer bind each other at the C-terminal capping domain, which accounts for dimer formation. The overall structure of mTLR3-ECD does not change upon binding to dsRNA, supporting a signaling mechanism in which ligand-induced receptor dimerization brings the two cytoplasmic TIR domains into contact, thus triggering a downstream signaling cascade. The dsRNA in the complex retains a typical A-DNA-like structure, in which the ribose-phosphate backbone and the position of the grooves are the major determinants of binding. The mTLR3-ECD interacts with the sugar-phosphate backbones, but not with individual bases. This explains why TLR3 lacks specificity for any particular nucleotide sequence. Point mutation in any one of the binding sites abrogate TLR3 function, indicating a mechanism in which three low affinity sites act cooperatively to form a stable signaling complex.